G-CSF (Granulocyte-Colony Stimulating Factor) is a naturally occurring growth factor which belongs to the family of cytokines. G-CSF plays a crucial role in hematopoesis and enhances maturation, proliferation, differentiation and survival of neutrophils and neutrophilic successor cells. Clinically, G-CSF is mainly used for controlling tumors and, in particular, for the treatment of neutropenia following chemotherapy, and it is also applied for bone marrow transplantations and in the treatment of infectious diseases.
Human G-CSF in its naturally occurring form is an about 20 kDa glycoprotein which has five cysteine residues. Four of these residues form two intramolecular disulfide bridges which are crucial for the activity of the protein. As G-CSF is obtained only in small amounts from natural sources, mainly recombinant forms of G-CSF are used in medicaments, in particular those which have been produced by expressing the protein in prokaryotic hosts. Proteins expressed in prokaryotic hosts such as E. coli differ from natural occurring G-CSF in that they are not glycosylated. Proteins expressed in E. coli have an additional N-terminal methionine residue necessary for expression in this host organism.
Due to the high hydrophobicity of the protein, non-glycosylated recombinant G-CSF is relatively unstable. The molecule easily adsorbs to the inner surface of storage vessels, vials, syringes or the like and forms dimers and higher aggregates. Conventional liquid G-CSF formulations also are sensitive to mechanical stress, for example as a result of shaking during transport, and to accidental freezing and thawing, which may also result in higher levels of aggregates and loss of biological activity. Moreover, G-CSF is subject to chemical modifications such as deamidation, oxidation, cleavage of disulfide bridges or proteolysis. Deamidation, which occurs more rapidly than other degradation routes, is a particular problem due to the high glutamine content of G-CSF. Altogether, this may result in a reduced content of biologically available and active monomeric G-CSF, particularly upon prolonged storage of the protein. This is not only costly but also is undesirable for therapeutic reasons, for example if the G-CSF is to be administered over a prolonged period of time at a constant dosage. Furthermore, products formed by multimerization or deamidation may result in an undesired immune response.
Stabilization of G-CSF formulations is subject of various patent and non-patent literature.
DE-A-37 23 781 describes aqueous phosphate-buffered G-CSF formulations containing pharmaceutically acceptable surfactants such as polyoxyethylene sorbitan esters which are used in combination with human serum albumin and mannitol for stabilizing the active ingredient. These formulations are stable at 4° C. over a prolonged period of time. Due to their antigenic properties, however, proteins and peptides of human and animal origin may cause undesired immunological reactions.
EP-A-0 373 679 discloses G-CSF formulations having a pH value of from 2.75 to 4.0 and low conductivity, which may be stored over prolonged periods of time without formation of aggregates. If any, buffer is used in these formulations in small amounts of less than 2 mM in order to avoid the aggregation of G-CSF.
EP-A-1 197 221 discloses long-term stable G-CSF formulations at a pH of from 5 to 7, which contain one or more amino acids of the group of lysine, histidine, arginine, aspartic acid, glutamic acid, threonine and asparagine, as well as one or more hydrophobic amino acids. Methionine is added to prevent oxidation of methionine residues in the G-CSF molecule.
WO-A-2007/034509 discloses stable aqueous formulations containing recombinant human G-CSF and an amino acid which is an oxidation suppressant for the methionine residues in the protein.
WO-A-2005/042024 discloses pharmaceutical compositions comprising G-CSF and an acid such as acetic acid or glutamic acid, which is free of surfactants.
WO-A-2005/039620 discloses succinate- and tartrate-buffered compositions stable over a wide pH range.
Herman, A. C. et al. (“Characterisation, Formulation, and Stability of Neupogen® (Filgrastim), a Recombinant Human Granulocyte-Colony Stimulating Factor.” In: Formulation Characterisation and Stability of Protein Drugs, pp. 303-328, R. Pearlman and Y. J. Wang, Eds., Plenum Press, New York, 1996) describe stabilized compositions of non-glycosylated recombinant G-CSF which contain 10 mM of sodium acetate, pH 4.0, 5% of mannitol and 0.004% of Polysorbate 80. Such compositions are stable for more than 24 months at 2-8° C. Substituting mannitol with sorbitol in a filgrastim formulation was found to eliminate sensitivity of the protein to aggregation during inadvertent freezing and thawing. Storing in a freezer, however, is to be avoided according to the manufacturer's instructions.
WO-A-2007/099145 discloses liquid acetate-buffered G-CSF formulations comprising polysorbate 20 and/or polysorbate 80 as a surfactant and having a pH-value between 4.1 and 4.4
WO-A-2008/122415 discloses liquid aqueous glutamate-buffered G-CSF formulations having a pH of from 3.5 to 4.8 which are stable under conditions of mechanical stress encountered, for example, upon freezing and thawing.